In the process of oxidative phosphorylation, the least understood event is the mechanism by which the redox processes are coupled to the synthesis of ATP. It is this enigma that has led to the postulation of the various theories of oxidative phosphorylation. The three major theories suggest that the redox energy is transformed into (a) a pH gradient, (b) a high energy state of the membrane, or (c) a chemical intermediate of high chemical energy. Photo-affinity labeling of mitochondria by an uncoupler may help differentiate among the above possibilities and clarify the mechanism of oxidative phosphorylation. The important questions potentially answerable by photo-affinity labeling are: 1. Which membrane components specifically interact with uncouplers? 2. Is there a finite number of binding sites for uncouplers? 3. Does uncoupling require the presence of freely mobile uncoupler molecules? We have synthesized a radioactively labeled water-soluble uncoupler of the dinitrophenol type. It has the structural requirements for a photo-affinity label, and it appears to bind to the oxidative phosphorylation system in a stoichiometric fashion. It is proposed to study the mechanism of uncoupling of oxidative phosphorylation using this and similar uncouplers. Uncoupler binding sites labeled by radioactive uncouplers will afford the isolation of the mitochondrial components involved in coupling oxidation to phosphorylation.